The overall aim of this proposal is to develop a 96-well plate screening assay that will allow screening for small molecule inhibitors of the cystine-glutamate antiporter, also known as system Xc-. System Xc- is a Na+-independent amino acid transporter that couples the export of intracellular L-glutamate with the import of extracellular L cystine. Cystine transport is critical for glutathione synthesis, an intracellular antioxidant whic protects cells from reactive oxygen species. Decreased glutathione levels lead to cellular growth arrest which has made system Xc- a target in cancer therapy, including brain cancer. In brain cancers, the release of glutamate via the antiporter raises additional concerns, as high levels of extracellular glutamate leads to peritumoral neuronal cell death aiding in tumor expansion by clearing surrounding tissue space. In addition to brain cancer, excessive glutamate release via system Xc- has also been shown to play a significant role in several neurodegenerative diseases wherein excess glutamate is presumed pathogenic, including Parkinson's disease, Alzheimer's disease, Multiple Sclerosis and epilepsy, which makes system Xc- a broad therapeutic target for neurodegenerative disorders. There are no selective and potent small molecule system Xc- inhibitors available to the public, and to our knowledge no high throughput screening for this target has been performed. As such, we propose to develop an HTS-ready 96-well assay to screen for novel chemical entities that inhibit system Xc- activity. We will validate this assay by screening four chemical libraries composed of clinically approved drugs as well as non-clinical compounds. The hits will be confirmed in a secondary screening assay and active novel compounds will be moved into structure activity relationship (SAR) studies guided by Xc- potency and in vitro drug- ability assays of metabolism, toxicity and target selectivity. As a future goal, lead compounds arising from these studies will be tested in animal models of glioma and multiple sclerosis and the validated 96-well assay will be further miniaturized to a 384-well format for high throughput screening of the NIH's >300 K compound library. PUBLIC HEALTH RELEVANCE: System Xc- is up-regulated in a variety of cancers and neurodegenerative diseases, and its inhibition with prototype small molecules or genetic deletion inhibits tumor growth in vivo and provides protection in animal models of neurodegeneration. There are no selective and potent small molecule system Xc- inhibitors modulators available to the public, and to our knowledge no high throughput screening (HTS) for this target has been performed. As such, we propose to develop an HTS-ready 96-well assay, screen multiple chemical libraries, and advance hits into a structure activity relationship (SAR) studies guided by Xc- potency and in vitro drug-ability assays of metabolism, toxicity and target selectivity.